This proposal describes a 5 year academic training program for a clinician-scientist. The candidate has completed almost a decade in General Surgery followed by a specialty fellowship in Pediatric Surgery. Due to a lengthy uninterrupted clinical track, the investigator proposes to complement prior training in immunology with current molecular techniques in gene therapy. This proposal will investigate the barriers to recombinant adeno-associated viral (rAAV) gene therapy and focus on abrogating host immune responses that may critically limit the effectiveness of gene transfer. Cystic fibrosis, CF, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) leading to dysfunction of the chloride channel, is the most common lethal autosomal recessive disorder in the Caucasian population and the primary reason for pediatric lung transplantation. Over the last decade significant progress has been made in the design of rAAV2 vectors as therapeutic vehicles to deliver a normal copy of the CFTR gene cDNA to the respiratory tract of CF patients with successful gene transfer and expression. Multiple challenges remain to enhance vector efficiency, deliver sufficient titers in situ, and precondition or modulate the host's microenvironment. Our recent work demonstrates that an endobronchial single administration of aerosolized rAAV2 vector can achieve high levels of gene transfer. However repeated dosing is most likely required for lifetime correction of CFTR dysfunction. An overall hypothesis is that the modulation of the local host immune response to a viral vector will lead to enhanced transgene expression and successful gene transfer upon repeated dosing. This hypothesis will be tested in the following specific aims, 1) to enhance gene transfer with an alternative serotype, AAV5, with an apical receptor ideal for aerosolized delivery. Investigating alternative serotypes to augment gene transfer efficiency and expression provides a scientific basis to select a serotype ideal for a given mode of delivery and target organ and to alternate serotypes to minimize potential immunization upon vector reexposures. Subsequent specific aims alter the host via immune modulation during the transient period of viral shedding, both 1) systemically and 2) locally in the pulmonary microenvironment to augment gene transfer. Studies of the immunobiology of rAAV vector system are crucial in order to overcome the immunologic barriers to effective gene therapies and limitations of single dose gene transfer for critical advance for a vector system with broad applicability. The laboratory of Dr. Guggino, the director of the Gene Therapy Center and an established investigator in the field, provides an ideal multidisciplinary environment with expertise in gene therapy and CFTR physiology, integrated in the setting of ongoing institutional CF clinical trials. The Department of Surgery provides the academic framework for clinician scientists to focus on establishing an investigatory career with novel therapeutics that potentiate the frontiers of pediatric surgery such as in utero intervention and/or surgery for congenital defects and in born errors of metabolism.